Genetic Analysis Using Genomic Representations

Proc. Natl. Acad. Sci. USA Vol. 95, pp. 4487–4492, April 1998 Genetics

Genetic analysis using genomic representations

ROBERT LUCITO*, MARIKO NAKIMURA*, JOSEPH A. WEST*, YING HAN*, KOEI CHIN†,KENDALL JENSEN*, RICHARD MCCOMBIE*, JOE W. GRAY†, AND MICHAEL WIGLER*‡

*Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724; and †Department of Laboratory Medicine, University of California, San Francisco, Box 0808 MCB 230, San Francisco, CA 94143-0808

Contributed by Michael Wigler, February 3, 1998

ABSTRACT Analysis of the genetic changes in human RBgl-24 [as used in Lisitsyn et al. (1)], oligonucleotides used tumors is often problematical because of the presence of for PCR of World-Wide Web probes, and tetranucleotide normal stroma and the limited availability of pure tumor repeat D17S695 were synthesized by BioSynthesis (Lewisville, DNA. However, large amounts of highly reproducible ‘‘repre- TX). Genescreen Plus was purchased from DuPont. Radioac- sentations’’ of tumor and normal genomes can be made by tive nucleotides, rediprime labeling kit, dNTPs, and hyperfilm PCR from nanogram amounts of restriction endonuclease were purchased from Amersham Pharmacia Biotech (Uppsala, cleaved DNA that has been ligated to oligonucleotide adaptors. Sweden). Cell lines used were obtained through American We show here that representations are useful for many types Type Culture Collection, grown in culture, and DNA- of genetic analyses, including measuring relative gene copy prepared. Normal human placenta DNA was obtained from number, loss of heterozygosity, and comparative genomic CLONTECH. Oligonucleotides and probes for use with the hybridization. Representations may be prepared even from Applied Biosystems 7700 Sequence Detector were synthesized sorted nuclei from fixed and archived tumor biopsies. by Applied Biosystems. Nusieve agarose gels used for analysis of PCR products were purchased from FMC. World-Wide Analysis of the genetic changes in human tumors is often Web probe sequences were downloaded from the Massachu- problematical because of the presence of normal stroma. setts Institute of Technology human genome sequencing da- Although either microdissection or flow cytometry can pro- tabase (http:͞͞www-genome.wi.mit.edu͞). duce small samples highly enriched for tumor cells or nuclei, Production of Representations. Genomic DNA (3–10 ng) the extracted DNA is of insufficient quantity for most uses. was digested by the desired restriction endonucleases (DpnII Nevertheless, we have successfully performed a complex pro- and BglII) under conditions suggested by the supplier. The tocol, representational difference analysis (RDA), on such digest was purified by phenol extraction and then precipitated small samples. RDA is a subtractive DNA hybridization tech- in the presence of 10 ␮g of tRNA. The digested DNA was nique that discovers the differences between paired normal resuspended by the addition of 444 pmol of each adaptor and tumor genomes (1). The first step of RDA is the prepa- (RBgl24 and RBgl12), T4 DNA ligase buffer (diluted to 1ϫ, ration of ‘‘representations,’’ which are highly reproducible provided with enzyme), and water to bring the volume to 30 ␮l. reformattings and amplifications of DNA populations. Typi- The reaction was placed in a 55°C heat block, and the cally, a representation is a set of restriction endonuclease temperature was decreased slowly to 15°C by placing the heat fragments of a limited size range amplified by PCR. As much Ϸ ␮ block at 4°C (for 1 hr). On reaching 15°C, the RBgl24 as 100 g of DNA can be prepared from as little as 3 ng of adaptor was ligated by the addition of 400 units of T4 DNA Ϸ ϫ 3 DNA ( 1 10 cells). ligase and by incubation at 15°C for 12–18 hr. The ligated In RDA, a representation with much lower complexity than material was divided into two tubes, and the following was the starting population is needed to enable a subtractive added: 80 ␮lof5ϫ PCR buffer [335 mM Tris⅐HCl, pH 8.8͞20 hybridization step to proceed effectively. Such low complexity ͞ ͞ ␤ ͞ mM MgCl2 80 mM (NH4)2SO4 50 mM -mercaptoethanol representations (LCRs) do not ‘‘capture’’ enough (typically, Ј Ј Յ 0.5 mg/ml of BSA], 2 -deoxynucleoside 5 -triphosphates to a 7%) of the genome to be useful for many of the more final concentration of 0.32 mM, RBgl24 adaptor to a final common types of analyses. However, we demonstrate here that ␮ concentration of 0.6 M, and H2O to bring the volume to 400 high complexity representations (HCRs) can provide ample ␮l. Each reaction was overlaid with 100 ␮l of mineral oil. The amounts of DNA in a sufficiently reproducible manner suit- reaction was placed in a thermal cycler preheated at 72°C, and able for most conventional studies. We demonstrate one type 15 units of AmpliTaq was added to the tubes. The thermal of HCR that captures Ϸ70% of the genome and illustrate its cycler was set to continue at 72°C for 5 min to allow for filling use for determining gene copy number, deletion mapping, loss in the 3Ј ends of the ligated molecules. This step was followed of heterozygosity (LOH), and comparative genomic hybrid- by 20 cycles lasting 1 min at 95°C and 3 min at 72°C, with an ization (CGH). HCRs may be a generally useful means of ‘‘immortalizing’’ and archiving DNA for later analysis from additional extension of 10 min at 72°C after the last cycle. The nonrenewable sources. PCR was divided into two tubes (now a total of four tubes), and the following reagents were added: 40 ␮l5ϫ PCR buffer (as above), dNTP to a final concentration of 0.32 mM, RBgl24 MATERIALS AND METHODS adaptor to a final concentration of 0.6 ␮M, water to bring the ␮ ␮ Materials. Restriction endonucleases as well as T4 DNA volume to 400 l, and 100 l of mineral oil. The tubes then ligase, T4 DNA polymerase, and T4 polynucleotide kinase were amplified for an additional five cycles with extension were supplied by New England Biolabs. AmpliTaq was sup- according to the above conditions. The reactions were purified plied by Perkin–Elmer. Oligonucleotide adaptors RBgl-12 and by phenol-chloroform and then precipitated by the addition of

The publication costs of this article were defrayed in part by page charge Abbreviations: HCR, high complexity representation; LCR, low complexity representation; RDA, representational difference analysis; CGH, payment. This article must therefore be hereby marked ‘‘advertisement’’ in comparative genomic hybridization; LOH, loss of heterozygosity. accordance with 18 U.S.C. §1734 solely to indicate this fact. ‡To whom reprint requests should be addressed at: Cold Spring © 1998 by The National Academy of Sciences 0027-8424͞98͞954487-6$2.00͞0 Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY PNAS is available online at http:͞͞www.pnas.org. 11724. e-mail: [email protected].

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1͞10th the reaction volume of sodium acetate (3M pH 5.2) and CGH. CGH was performed according to standard proce- by the addition of one reaction volume of isopropanol. dure (9) by using HCR DNAs and genomic DNAs prepared PTEN Tumor Suppressor Lori Genomic Sequencing. Bac- from BT474 and MCF7 as target and normal female human terial artificial chromosome DNA was purified by detergent lymphocyte as the reference. The genomic and HCR DNA lysis and polyethylene glycol precipitation. DNA (5 ␮g) was samples were labeled by nick-translation with fluorescein sheared and then repaired by using T4 DNA polymerase. isothiocyanate–12-dUTP and Texas Red 5-dUTP to produce Fragments in the range of 1.5–2 kb were isolated by gel DNA fragments ranging in size between 500 and 2,000 bp. fractionation and inserted into M13 by ligation. Sequencing Quantitative PCR. Primer and probe sequences were pro- reactions were done by using dye primer chemistry that uses duced according to the Applied Biosystems software PRIMER energy transfer primers (2) and thermosequenase polymerase EXPRESS. These primers and probes were used in a PCR that (3). Reactions were run on an Applied Biosystems 377 se- was placed in the Applied Biosystems 7700 Sequence Detector quencer for 6- to 8-hr sequence runs for analysis. Base calling and amplified as described (10, 11). Samples used were HCRs was carried out by using the program PHRED (P. Green, produced from DNA derived from nuclei of sorted primary University of Washington). The collection of initial data was tumor biopsies. After the PCR, the data of fluorescence assembled into a contig by using the automated assembly corresponding to cycle number were downloaded from the program PHRAP (P. Green). The database generated was Applied Biosystems 7700 Sequence Detector to Microsoft converted to XGAP format and edited by using XGAP (4, 5). EXCEL and analyzed to give the graphs shown later. After completion of Ϸ6-fold coverage, the finishing process was carried out by using the program FINISH (G. Marth, RESULTS Genome Sequencing Center at Washington University) to fill gaps in the contig. The Basic Method. DNA from samples, usually paired tumor Southern Blotting. Normal DNA from a tumor cell line or cells or nuclei and normal cells or nuclei, were processed in representations derived from sorted nuclei were digested with parallel to prepare representations. DNA was cleaved with a either DpnII or BglII. Digested genomic DNA (5.0 ␮g), restriction endonuclease, such as DpnII or BglII, that is not digested HCR (5.0 ␮g) , or digested LCR (0.5 ␮g) was loaded blocked by 5-methylcytosine, and double-stranded, cohesive, on a 1.5% agarose gel. The gel was transferred to Genescreen adaptor oligonucleotides were ligated to the fragment ends. Plus after electrophoresis was completed. After transfer, the The adaptors were not phosphorylated and therefore could not blot was hybridized in GIBCO prehybridization solution at be self-ligated to form interfering dimers. Because adaptors 67°C for 2 hr. The probe was produced by random priming with can be ligated only to the 5Ј ends of the cleavage fragments, the the rediprime kit in the presence of 32P␣dCTP. After 12–18 hr ligated product then was treated with DNA polymerase to fill of hybridization, the blots were washed with standard saline in the 3Ј ends, forming the primer binding site for the phosphate͞EDTA buffer as described (6). The blots were used subsequent PCR. Amplification was performed in stages, each for producing either auto-radiograms and͞or phosphorimages. stage using the adaptor oligonucleotides as primers and pro- LOH Analysis. LOH was assessed by using the tetranucle- ceeding for no more than 20 rounds per stage. PCR does not otide repeat marker D17S695 (listed as UT269 in the National amplify all fragments equally well, and high molecular weight Center for Biotechnology Information Web Site http:͞͞ fragments in particular are very poorly amplified. Thus, re- www.ncbi.nlm.nih.gov͞Entrez͞nucleotide.html), which maps striction endonucleases that cleave infrequently were used to to the 17p13 region of the human genome and has been used prepare LCRs, and enzymes that cleave frequently were used to determine the state of LOH at the p53 locus (7, 8). The to prepare HCRs (see Fig. 1). From 3 ng of starting material, reverse primer was labeled by phosphorylation in the presence we could obtain 40 ␮g of total HCR from a total of 25 rounds. of 32P␥ATP as described (7, 8). The labeled reverse primer in We have not tested the usefulness of an HCR beyond a total combination with both forward and reverse primers was used of 35 rounds. for PCR by using 50 ng of HCR as DNA template. PCRs were Sampling the Complexity and Reproducibility of DpnII performed as described in the National Center for Biotech- Representations. We first sampled the reproducibility and nology Information Web Site, and gel electrophoresis was complexity of DpnII HCRs. We analyzed 14 different HCRs, performed as described (7, 8). each made from 5 ng of DNA prepared from diploid nuclei

FIG. 1. Schematic comparison of LCR and HCR, illustrating the complexity reduction that occurs after cleavage with rare and frequent cutters, respectively. Downloaded by guest on October 1, 2021 Genetics: Lucito et al. Proc. Natl. Acad. Sci. USA 95 (1998) 4489

separated from tumor biopsies by flow cytometry and each amplified by PCR for 25 rounds. In our first sampling, we designed pairs of PCR primers to detect Web Site sequence- tagged sites. We picked sequence-tagged sites that were not cleaved by DpnII and used primer pairs that amplified a single band from total genomic DNA controls. Of these, 18 of 25 pairs (72%) were able to amplify the same molecular weight fragment from each HCR, and 7 generally failed to amplify from any HCR. Our results suggest that DpnII HCRs repro- ducibly contain the same elements and Ϸ70% of the genome. We performed a similar sampling with primer pairs derived from the locus encoding the PTEN tumor suppressor gene, for which we had the complete nucleotide sequence (unpublished data). In this way, we were able to use primers derived from DpnII fragments of known size. DpnII fragments were chosen at random, and PCR primer pairs were designed for each. Primer pairs (22 pairs) amplified single fragments by PCR from control genomic DNAs. These pairs were used with the same panel of 14 HCRs used above. Primer pairs (20 pairs) amplified the expected fragment from all HCRs, and 2 pairs failed to amplify from any. The fragments that were not in the HCRs were the largest, 3,916 bp, and one of the smallest, 97 bp. Totaling all fragment lengths, 16,039 bp were included in the HCRs, and 4,013 bp were excluded. Thus, assuming that our initial selection of DpnII fragments was random, the HCRs contained Ϸ75% of the PTEN region. If DpnII cleavage is nearly complete during the preparation of an HCR, we expect that no PCR primer pairs should readily amplify from an HCR when the amplified sequence has an internal DpnII site. To test this, we chose four primer pairs from the PTEN locus that amplified a single fragment containing a single internal DpnII site. All four pairs amplified fragments from genomic DNA controls, and none amplified detectable fragments from the 14 HCRs. We conclude that HCRs prepared in parallel from samples processed in parallel are reasonably reproducible and represent Ϸ70% of the human genome. Measuring Gene Copy Number in HCRs. Tumor genomes often contain either extra copies of sequences caused by gene amplification or missing sequences caused by gene deletion. To FIG. 2. Analysis of copy number using representations (Rep) and explore the usefulness of representations for measuring gene genomic DNA (Gen) for c-erbB2 and c-myc. (A) Southern blot copy number, we first compared Southern blots of genomic comparing tumor cell lines (T) to normal (N) cell lines. DpnII was used DNA to blots of HCRs and LCRs. For this purpose, we used to prepare HCRs (DpnII), and BglII was used to prepare LCRs (BglII). human placental DNA as normal and prepared genomic DNA As a reference, free probe also was run alongside the samples (probe). from tumor cell lines amplified at c-erbB2 (BT-474) (12) or The hybridization probes were derived from small BglII fragments c-myc (SK-BR-3) (13). HCRs and LCRs were made from cell isolated from P1 clones specific for each locus respectively. (B) Quantitation of the Southern blot. Rep, representation; Gen, genomic line or placental DNAs by using DpnII or BglII, respectively. DNA; DpnII, either DpnII HCR or DpnII digest of genomic DNA; As probes we used small BglII fragments that we cloned from BglII, either BglII HCR or BglII digest of genomic DNA. Blots in A P1s containing inserts from the designated loci. The blots, were stripped and reprobed with a single copy probe to analyze loading shown in Fig. 2A, were quantitated by phosphorimaging. To differences (data not shown). Phosphorimage analysis of this probe normalize for loading differences, the blots were stripped and was used to normalize the amplification differences. After this anal- rehybridized with a single copy sequence probe. The normal- ysis, the resulting intensity of tumor was divided by the intensity of ized ratios of signal from tumor and normal are tabulated in normal to give the fold amplification displayed. (C) Deletion mapping Fig. 2B. The same relative copy number (tumor to normal) was using HCRs and the deletion mapping of seven tumor cell lines determined from blots of representations as was determined comparing blots of HCRs (HCR) with blots of the corresponding genomic DNA (Genomic), each designated 1–7. The probe used for from blots of genomic DNAs, indicating that there is a hybridization is from the human genomic region 20p11 (HCR). The quantitatively reproducible amplification of these test se- Genomic panel shows the same DNAs blotted with an unrelated probe. quences during the preparation of either HCRs or LCRs prepared in parallel from similar starting materials. Similar We tested the value of the HCRs made from limited results were obtained for the cyclin D locus (data not shown). amounts of DNA for quantitation of copy number. HCRs To explore the usefulness of HCRs for deletion mapping, we blotted both genomic and HCR DNAs from tumor cell lines prepared from aneuploid and diploid nuclei sorted from for deletion at the 20p11 locus. This locus was discovered several breast cancer biopsies were blotted for c-erbB2. Fig. 3A initially by using RDA and subsequently was found to be illustrates that c-erbB2 is amplified in the HCRs made from the deleted frequently in gastrointestinal cancers (R.L., unpub- aneuploid nuclei of some biopsy samples. Fig. 3B shows the lished data). Fig. 2C illustrates that the probe hybridized to blots with an unrelated probe. We obtained confirmation of sequences in the HCRs when and only when it hybridized to the validity of the c-erbB2 amplifications by demonstrating sequences in the respective genomic DNA. An unrelated probe that probes adjacent to but distinct from the c-erbB2 probe also detects sequences present in all genomic samples and their were amplified in the same samples (M.N., unpublished re- representations. sults). Downloaded by guest on October 1, 2021 4490 Genetics: Lucito et al. Proc. Natl. Acad. Sci. USA 95 (1998)

one-eighth as much in the aneuploid HCR), probably reflect- ing Ϸ10% contamination of the aneuploid nuclei with diploid nuclei after sorting. These results were confirmed by Southern blotting (data not shown). One tumor͞normal pair showed a shift of a single cycle for primer pairs detecting the p16 gene, which might reflect loss of a single allele in the tumor or experimental error. Detection of LOH in HCRs. LOH is a common lesion found in cancer cells and may be indicative of genomic instability and͞or the loss of function of a specific tumor suppressor gene FIG. 3. Comparison of HCRs from primary tumor biopsies by (14–17). The detection of LOH often is obscured by the Southern blotting. (A) HCRs from the designated tumor biopsies presence of normal stroma; hence, we tested whether HCRs (denoted by an identification number preceded by BBR) were pre- prepared from minute amounts of samples highly enriched for pared from diploid (Dpl) and aneuploid (Anu) nuclei and compared tumor nuclei could be used for LOH analysis. PCR primers by Southern blot analysis. The c-erbB2 probe was the same as that used in Fig. 2. (B) The same DNAs blotted with an unrelated probe. that amplify microsatellites and detect fragment length poly- morphisms are used frequently for LOH mapping, and we Finally, we tested some of the same samples by quantitative chose to examine a primer pair that amplifies a highly poly- PCR. For this purpose, we used the dual-labeled fluorogenic morphic tetranucleotide repeat near the p53 locus (7, 8). hybridization probes and the Applied Biosystems 7700 Se- In preliminary experiments, we established that these PCR quence Detector (10, 11) to compare HCR DNAs prepared primers detected the same allele pattern in both genomic and from aneuploid and diploid nuclei pairs. The results, shown in HCR DNAs prepared from cell lines. Next, we examined 12 Fig. 4, indicate that differences in copy number were detected pairs of HCRs prepared from aneuploid and diploid nuclei. by probes for the c-erbB2 oncogene and the p16 tumor LOH at this locus was detected clearly in 9 of 10 informative suppressor. On the other hand, no differences in copy number pairs (see Fig. 5 for representative cases), which is greater than were detected by probes from an uninvolved region on chro- the reported proportion of LOH at this locus in breast cancer mosome 3. The curve of amplification of the c-erbB2 fragment (50%) (14, 18, 19) but may reflect the selection of the highly arises four cycles sooner in one aneuploid HCR than it does in aneuploid tumors that we sorted and͞or the purity of our the paired diploid HCR, indicating a higher copy number for samples. c-erbB2 (Ϸ16-fold higher in the aneuploid HCR sample). The Comparative Genome Hybridization with HCRs. CGH is a curve for the aneuploid HCR deleted for p16 arises three powerful tool for analyzing the global genomic changes of cycles later than the paired diploid HCR (approximately tumors (20–25). It has been reported that chromosome paint-

FIG. 4. Quanitative PCR analysis of HCRs. Diploid (black) and aneuploid (gray) HCRs derived from sorted primary tumor biopsies were used as template for quanitative PCR analysis. Probes from several genomic loci (FHIT, p16, and c-erbB2) were used to determine copy number in three primary tumor pair HCRs (BBR44, BBR49, and CHTN5). The data from the Applied Biosystems 7700 Sequence Detector was analyzed with Microsoft EXCEL to produce the graphs shown. (x axis, the cycle number during the reaction; y axis, the fluorescence detected.) Downloaded by guest on October 1, 2021 Genetics: Lucito et al. Proc. Natl. Acad. Sci. USA 95 (1998) 4491

ducibility of the representation, and the loss of natural restriction sites at the ends of the amplified material make the usefulness of this method somewhat limited. Restriction endonuclease-based representations have major advantages: Their complexity can be regulated by the choice of restriction enzyme; they can be readily reamplified; they can be analyzed by Southern blotting; and they are highly reproducible. In this report, we have explored the uses of HCRs, including quantitative assessment of copy number, LOH, and CGH. All FIG. 5. Using HCRs for LOH analysis. LOH analysis was carried out on HCRs derived from sorted primary tumor biopsies. Dpl, diploid of these analytical methods require a high level of reproduc- HCR; Anu, aneuploid HCR. The tumors are labeled as either BBR, ibility in the representation. To achieve this level of reproduc- CHTN, or NSBR followed by an identification number. The primers ibility, we have prepared paired samples of HCRs from the used in the reaction amplify a tetranucleotide repeat within the p53 same amount of starting material, used genomic DNAs ex- locus. A mixed population of normal DNAs was used as a positive tracted in the same manner, and performed PCR at the same control. (ϩGen, normal genomic DNA used as template; ϩHCR, time, under the same conditions, and in the same thermal HCR produced from this normal DNA used as template. Ϫ, reaction cycler. in which no template was added.) In principle, HCRs can be prepared from normal and tumor tissue stored as fixed, paraffin-embedded, archived biopsies, ing could be performed with representations produced from which would extend greatly the usefulness of such samples. We each chromosome (26). We therefore tested whether CGH have made HCRs from DNAs extracted from pairs of aneu- could be performed with HCRs. For this experiment, we chose ploid and diploid nuclei sorted from such sources (R.L., to examine tumor cell lines so that we could directly compare unpublished data). More rounds of PCR are required to obtain CGH performed with genomic DNA with CGH performed workable amounts of DNA, and HCRs from DNA extracted with HCR. Little difference between HCR and genomic DNA from fixed specimens have a markedly lower size distribution could be discerned with either of the two cell lines examined, than HCRs prepared from fresh sources. Moreover, there is BT-474 and MCF7. Fig. 6 shows a sample of the chromosomal enormous variability between the HCRs from different spec- scanning profiles obtained with each DNA source. imens, reflected as varying size distributions. This diversity is probably caused by the variation in the quality of the DNA that DISCUSSION can be extracted from specimens fixed and stored under different conditions (19, 34–36). Despite the variability be- The idea of capturing essential features of the genome as a tween specimens, we found that the HCRs prepared from PCR product is not new. In 1989, Kinzler and Vogelstein (27) aneuploid and diploid nuclei from the same fixed specimen are described ‘‘whole genome’’ PCRs to select for DNA sequences similar to each other. Indeed, we have found that HCRs that were binding sites for DNA binding proteins. Their prepared from sorted nuclei of fixed specimens are useful for method was used in 1995 (28) for the same purpose. In the CGH (unpublished observations). These results suggest that experiments from 1989, PCR adaptors were blunt-end ligated normal and tumor HCRs will be useful even when prepared for to the cleavage fragments of total genomic DNA. The useful- analysis from microdissected specimens. ness of the resulting PCR products for genomic analysis was Both HCR and LCR render paired tumor and normal DNAs not explored; nor was its efficiency explored when starting with from minute specimens in a stable format that can be analyzed tiny amounts of material. An alternate approach to whole or further amplified at a later date. We have emphasized the genome PCR is to use random priming with degenerate usefulness of HCRs for gene copy, LOH, and global genomic oligonucleotides (29–33). This method can produce large analysis of tumor specimens. Most probes, chosen at random, amounts of DNA starting from very minute amounts of will be present in an HCR but will not be present in the LCRs. sample, but the complexity of the DNA produced, the repro- However, precisely because LCRs are of lower complexity,

FIG. 6. CGH analysis using HCRs. Shown are two representative chromosome spreads (Ch 1, and Ch17) comparing the genomic (Gen) with the HCR for two different cell lines, BT474, and MCF7. Deviation from the dashed line represents genomic change, a peak represents gain in copy number, and a trough represents loss of copy number. Underneath the profiles is an idiogram of each chromosome as a reference. Downloaded by guest on October 1, 2021 4492 Genetics: Lucito et al. Proc. Natl. Acad. Sci. USA 95 (1998)

hybridization-based assays that depend on completeness of 16. Wada, C., Shionoya, S., Fujino, Y., Tokuhiro, H., Akahoshi, T., hybridization are easier to perform. This ease is apparent in Uchida, T. & Ohtani, H. (1994) Blood 83, 3449–3456. blotting analysis of LCRs. For the same reason, some global 17. Wieland, I., Ammermuller, T., Bohm, M., Totzeck, B. & Rajew- genomic analyses, such as microchip array analysis (37–40) or sky, M. F. (1996) Oncol. Res. 8, 1–5. 18. Chen, L. C., Neubauer, A., Kurisu, W., Waldman, F. M., Ljung, CGH (20–25), that depend on hybridization kinetics should be B. M., Goodson, W., III, Goldman, E. S., Moore, D., II, Balazs, facilitated by the use of LCRs because reannealing times M., Liu, E., et al. (1991) Proc. Natl. Acad. Sci. USA 88, 3847–3851. should be reduced and signal-to-noise ratios enhanced. 19. Chen, Y. H., Li, C. D., Yap, E. P. & McGee, J. O. (1995) J. Pathol. 177, 129–134. We thank Masaaki Hamaguchi, Eli Hatchwell, and Clifford Yen for 20. 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